Supplemented Serum-Containing Culture Medium for Enhanced Arpe-19 Growth and Human Cytomegalovirus Vaccine Production

Abstract

The present invention relates to supplemented serum-containing cell culture media that provides enhances ARPE-19 cell growth and/or improves the yield of human cytomegalovirus (HCMV) grown in ARPE-19 cell cultures. The media of the invention includes two additives, a hormone (e.g., a glucocorticoid hormone such as dexamethasone) and a growth factor (e.g., EGF). The invention further provides methods of producing HCMV in such growth media.

1 . A cell culture medium comprising:
 a basal cell culture medium comprising; a serum comprising between about 0.1 μM and about 10 μM of dexamethasone, and between about 0.1 ng/mL and about 100 ng/mL of epidermal growth factor (EGF).   
     
     
         2 . The medium of  claim 1 , wherein the basal cell culture medium is DMEM/F-12. 
     
     
         3 . The medium of  claim 1 , wherein said serum is fetal bovine serum (FBS). 
     
     
         4 . The medium of  claim 3 , wherein the FBS is present at a concentration between about 2% and about 10%, inclusive. 
     
     
         5 . The medium of  claim 1 , wherein the dexamethasone is present at a concentration between about 1 and about 10 μM. 
     
     
         6 . The medium of  claim 1 , wherein the EGF is present at a concentration between about 1 and about 40 ng/mL. 
     
     
         7 . The medium of  claim 1 , further comprising 1× MEM essential amino acids, wherein the 1× MEM essential amino acids consist of arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine. 
     
     
         8 . The medium of  claim 1 , further comprising: 1×-4× glutamine synthetase expression medium (GSEM) supplement, wherein the GSEM supplement consists of a mixture of L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, adenosine, guanosine, cytidine, uridine, and thymidine. 
     
     
         9 . The medium of  claim 1 , further comprising trace elements, wherein the trace elements are selected from cupric sulfate, ferric citrate, sodium selenite, and zinc sulfate. 
     
     
         10 . A composition comprising the medium of  claim 1  and cells selected from ARPE-19, Vero, MDCK, MRC-5, BHK, CEM, and LL-I cells. 
     
     
         11 . The composition of  claim 10 , wherein said composition further comprises a virus. 
     
     
         12 . The composition of  claim 11 , wherein said virus is a human herpesvirus. 
     
     
         13 . The composition of  claim 12 , wherein said human herpesvirus is human cytomegalovirus (HCMV). 
     
     
         14 . The composition of  claim 13 , wherein said HCMV is a conditional replication defective HCMV mutant. 
     
     
         15 . The composition of  claim 14 , wherein the conditional replication defective HCMV mutant has a genomic sequence of SEQ ID NO: 1. 
     
     
         16 . A method of increasing virus production in cells, said method comprising culturing the composition of  claim 10 , wherein said composition provides increased production of the virus by at least 10% relative to the production of virus grown in a composition that does not contain the supplements of  claim 1 . 
     
     
         17 . A method of increasing virus production in cells comprising culturing a cell culture medium comprising:
 a basal cell culture medium comprising:   a serum comprising dexamethasone in the amount of about 0.1 μM to about 10 μM; epidermal growth factor (EGF) in the amount of about 0.1 ng/mL to about 100 ng/mL; wherein the composition provides increased production of the virus by at least 10% relative to the production of virus grown in a composition that does not include the basal cell culture.   
     
     
         18 . The method of  claim 17 , wherein the serum further comprises 1× MEM essential amino acids, wherein the 1× MEM essential amino acids consist of arginine, cysteine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine. 
     
     
         19 . The method of  claim 17 , wherein the serum further comprises 1×-4× glutamine synthetase expression medium (GSEM) supplement, wherein the GSEM supplement consists of a mixture of L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, adenosine, guanosine, cytidine, uridine, and thymidine. 
     
     
         20 . The method of  claim 17 , wherein the serum further comprises trace elements, wherein the trace elements are selected from cupric sulfate, ferric citrate, sodium selenite, and zinc sulfate.